Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9)

Objectives: While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ± I222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness.

Methods: Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ± I222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, K_m of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined.

Results: NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA.

Conclusions: NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued.